Flavobacterium psychrophilum is a Gram-negative bacterial fish pathogen that causes bacterial coldwater disease (CWD) and is considered to be one of the most important pathogens affecting salmonid aquaculture due to its wide distribution and economic impact. In the United States, it is estimated that annual losses incurred from CWD in the Pacific Northwest alone are approximately 9.6 and 4 million dollars for commercial aquaculture of rainbow trout (Oncorhynchus mykiss Walbaum) and conservation aquaculture of salmonid species, respectively.
Preventative measures include the use of management strategies to reduce risk factors such as stress, poor water quality, and cutaneous lesions. Even with these in place, CWD commonly occurs and generally requires treatment. Treatment options are limited and include reducing pathogen concentrations, eliminating the spread of the pathogen, and the use of antibiotics. However, the effectiveness of treatment is usually inconsistent, and there are potential risks of developing antibiotic resistant strains. Therefore, a vaccine to prevent CWD is desired. However, even though the need is great and has long been sought, no vaccines for CWD are currently available.
In order to be commercially useful, a vaccine for fish must be capable of conferring protective immunity against a pathogen when the vaccine is administered by practical methods, such as immersing the fish in water containing the vaccine. Vaccination protocols that require individual handling of fish, such as by injection are not practical for most commercial aquaculture operations.
Immunization with killed bacteria has been attempted with F. psychrophilum, and protection obtained by immersion or by injection with the killed bacteria has been minimal.
Better protection has been obtained by administering the killed bacteria by injection in combination with an emulsified adjuvant. However, because such vaccination protocols require individual handling of fish, they are less suitable for most aquaculture applications.
Recently, live attenuated bacterial vaccines have been developed to immunize animals against particular diseases. Direct and random approaches can be used to induce mutations into bacterial pathogens to achieve attenuation. Direct approaches include mutation or deletion of genes involved in metabolic pathways and/or pathogenesis, while random approaches include genetic methods such as transposon mutagenesis or the use of chemicals such as antibiotics. In the latter method, bacteria are cultured on or in a medium containing a chemical compound that induces one or more non-lethal mutations in the bacteria, while maintaining the protective immunogenicity of the bacteria.
Antibiotics that promote mutations in bacteria have been found to be useful in the development of attenuated bacteria. The development of resistance to high concentrations of the antibiotic may be correlated with changes in the genotype or phenotype of the bacteria. Such changes are often associated with attenuation of the bacteria while maintaining the immunogenicity of the organism.
One such antibiotic that has been shown to be useful in creating attenuated bacteria is rifampicin. Rifampicin is a broad spectrum antibiotic that inhibits bacterial DNA-dependent RNA polymerase.
A rifampicin-resistant attenuated live vaccine was developed to protect cattle against the effects of infection with Brucella abortus. The development of this vaccine was reported in Schurig, Veterinary Microbiology, 28:171-181 (1991). As described in Schurig, the B. abortus organism was attenuated by passage of virulent strain 2308 numerous times on medium supplemented with increasing concentrations of rifampicin.
Rifampicin resistant bacteria have also been utilized in the development of attenuated bacterial vaccines for diseases affecting fish. Attenuated rifampicin-resistant live bacterial vaccines for diseases affecting fish are disclosed in Klesius, U.S. Pat. No. 6,019,981; Shoemaker, U.S. Pat. Nos. 6,881,412; and 6,991,793; and Evans, U.S. Pat. No. 7,067,122.
Klesius discloses an attenuated live bacterial vaccine against enteric septicemia of catfish caused by Edwardsiella ictaluri. The rifampicin-resistant bacteria were determined by SDS-PAGE not to produce the O-polysaccharide (O—PS) side chain component of lipopolysaccharide, which is accepted as an important virulence factor of this organism. Klesius discloses that the attenuated strains of E. ictaluri differentiated from the parent microorganism because they were resistant to rifampicin and that biochemical characteristics of the attenuated organisms were identical to those of the parent microorganism.
Shoemaker discloses an attenuated live bacterial vaccine against Flavobacterium columnare, the causative agent of columnaris disease. Shoemaker discloses that the attenuated strains of F. columnare differentiated from the parent microorganism because they were resistant to rifampicin and that biochemical characteristics of the attenuated organisms were identical to those of the parent microorganism.
Evans discloses an attenuated live bacterial vaccine against Edwardsiella tarda, the causative agent of Edwardsiella septicemia disease. Evans discloses that the attenuated strains of E. tarda differentiated from the parent microorganism because they were resistant to rifampicin and that biochemical characteristics of the attenuated organisms were identical to those of the parent microorganism.
Each of the attenuated live vaccines of Klesius, Shoemaker, and Evans was effective when administered to fish by immersion. The effectiveness of immersion vaccination with each of these three vaccines is not surprising because fish are readily infected with each of the three diseases for which the vaccines were developed; enteric septicemia, columnaris disease, and Edwardsiella septicemia, by immersion in water containing the causative organisms.
Coldwater disease, unlike the diseases disclosed in Klesius, Shoemaker, and Evans, cannot be effectively introduced into fish by immersion in the absence of some portal of entry. The present inventors have immersed salmonids into water containing high levels of the causative organism of CWD, F. psychrophilum, and have been unable to induce disease in this manner. However, if fish were exposed to F. psychrophilum by subcutaneous injection, infection with development of disease readily occurred. It is also known that, if fish are wounded prior to the immersion exposure, such as by a pinprick or removal of scales, infection with development of disease occurs.
In view of the fact that infection following exposure to F. psychrophilum by immersion does not occur in fish, it would not be expected that vaccination by immersion exposure to attenuated live F. psychrophilum would provide protective immunity against CWD.